Project Design Safety

Our project utilizes engineered MSCs to improve wound healing meanwhile addressing safety concerns related to YAP-1 expression, so we illustrate here the steps which we took to ensure the safety in our project design:

1. dCas9-synRTK receptor: a synthetic receptor composed of two main domains. Each domain consists of two separate longitudinal chains. To prevent any possible hazard, we were even concerned with implanting safety regulations in each domain.
   

   (Figure 1) Receptor's Structure

   1. In the extracellular domain: We targeted a specific tissue injury biomarker called vascular endothelial growth factor(VEGF) through VEGF - receptor1 in the first chain and VEGF - receptor 2 in the second chain. Each chain’s receptor has a different affinity to VEGF as VEGFR-2 will bind to VEGF. Then, this complex will bind to the VEGFR1 chain. Significantly, this different affinity results from structural difference in each receptor’s internal domain.
   2. In the Internal domain:The first chain, which carries VEGFR-2, consists of the N-domain of tobacco etch virus (TEV) protease, nuclear export signal (NES), TEV cleavage site(TCS), and the dcas-9 N-domain. On the other hand, the second chain is composed of the C-domain of TEV protease, TCS, nuclear localization signal (NLS), and the dcas-9 C-domain - loaded by transcriptional activator factors. These domains will unite and transfer to the nucleus under the guidance of NLS. In contrast, in absence of VEGF, the N-dcas-9 domain separates away from the C-dcas-9 domain under the guidance of NES ( extra-nuclear) and NLS (intra-nuclear), respectively.
   

   (Figure 2) Receptor Dimerization

2. mRNA Switch:Availing MSCs ability to communicate with cells, we took advantage of its exosomes by delivering our YAP-1 mRNA switch. It is meant to increase YAP-1 expression locally from viable surrounding cells. Initially, our mRNA is in a linear form and turns to be in a circular form when activated. We warranted the switch’s safety through:
1. We aimed to increase specificity through targeting an intracellular tissue injury biomarker called matrix metalloproteinase-9 (MMP-9). Therefore, we designed MMP-9 nanobody on both mRNA ends. On the poly A tail end, we added MMP-9 nanobody attached to MS-2 aptamers through MS-2 coat protein (MCP). Similarly, on the cap side, we attached MMP-9 nanobody on the cap through NSP3. When MMP-9 binds to its own specific sites, our mRNA becomes in an active circularized form.
2. Our mRNA undergoes a minimal basal activity, resulting from the poly A tail. However, this poly A tail plays a great role in the mRNA stability. Hence, to increase the stability and decrease the basal activity of our switch, we implemented a hammerhead ribozyme (HHR) just before the poly A tail. In MMP-9 existence, HHR goes through self cleavage to separate the poly A tail, producing an active circular controlled mRNA without a basal activity.


(Figure 3a) HHR self-cleavage



(Figure 3b) Active circular YAP-1 mRNA

At the end, our project design enables controllable gene regulation in response to specific environmental signals, promising significant advancements in synthetic biology applications.

Challenges

We faced many challenges during our journey:

Short half-life of MSCs

MSCs have high regenerative function and have low immunogenicity. It has a problem which is its short half-life especially when applied to wounds being exposed to an external environment where its lifespan average is 3 days . For this problem the solution was to enhance its regenerative function through increasing the endogenous expression of YAP-1 in the MSCs during its narrow span. Another solution was the administration of MSCs in hydrogel increasing its lifespan to about 7 days.

The carcinogenic effect of MSCs

Despite the various benefits of mesenchymal stem cells one of which was its regenerative function there was a point of highest concern to us which was its oncogenic risk when used as a therapy moreover the increased expression of YAP in the mesenchymal stem cells leading to further increase in the oncogenic risk. So, our savior in this problem was the dcas9-synRTK receptor which is bound by its internal domain to dCas9 (c) which won’t be released until dimerization of the receptor which occurs only when VEGF is present causing activation of the protease enzyme at the cleavage sites .Then, the dCas9(c) binds to the N-domain of dCas9 leading to expression of endogenous YAP by the MSCs.

Safety Switch

We had concern about the continuous expression of YAP in the genetic circuit once it is delivered to nearby keratinocytes so for regulation of the mRNA translation we made it activate by presence MMP9 molecule which bound to its specific nanobody present on the two ends of the causing loop shape of mRNA (translation activated) and once MMP9 disappears it will result in stopping the translation further preventing any carcinogenic complication.

Fig 2.

References

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